PROJECT SUMMARY Arthropod-borne viruses (arboviruses) are responsible for a significant disease burden worldwide. Zika virus (ZIKV) is the most recent arbovirus to emerge and threaten public health. Local mosquito transmission of ZIKV was first identified in the western hemisphere in Brazil in 2015. ZIKV exploded in this nave human population and spread to many countries in the Americas and Caribbean, where local mosquito transmission has resulted in millions of infections. Most alarming are the serious complications associated with ZIKV infection, including microcephalic infants born to ZIKV-infected mothers and Guillain-Barr syndrome, an autoimmune neurological disease. Despite recent research efforts, these complications and ZIKV pathogenesis in general are poorly understood, prompting the NIH to call for basic and translational research on ZIKV as a high priority (NOT-AI-16-026), including a special funding opportunity to provide research support on ZIKV and its complications (PA-17-085). In this proposal, we will directly address this high priority announcement and funding opportunity by focusing on the phenotypic characterization of novel adaptive ZIKV mutations, which have the potential to alter virulence, mosquito transmission, and/or emergence. In these studies, we will test our overall hypothesis that the adaptive mutations will increase virus growth kinetics and impact virulence and/or vector competence. Our proposed studies will contribute to the field by providing critical information on genetic determinants of ZIKV and will lay the foundation for future mechanistic studies in ZIKV biology and transmission.